Hypoxic cells in solid tumors are resistant to killing by ionizing radiation, and are thought to limit the success of radiotherapy. Many different methods have been designed to reduce or eliminate the hypoxic cell problem, yet no convenient or accurate method is currently available to actually measure hypoxic cells. Techniques to quantify cellular hypoxia are now being developed based primarily on the preferential metabolism and binding of fluorescent nitroheterocycles by hypoxic cells. Fluorscence- activated cell sorting (FACS) allows the fluorscence of individual cells to be measured, and this estimate of oxygenation can be correlated with viability measurements and biochemical analyses of cells sorted according to their flurorescence intensity. The feasibility of this method has been demonstrated in single cells and spheroids, and will now be extended to mouse tumor models. In addition, using the fluorescent probe AF-2, studies will continue on reasons for heterogeneity of AF-2 binding in multicell spheroids and tumors. New probes will be evaluated in vitro and in vivo, in the search for more fluorescent, less toxic nitroheterocycles, and for other fluorescent markers which may localize in hypoxic microregions in tumors. Finally, the roles of necrosis and transient hypoxia in binding of hypoxia probes in tumors will be addressed. In addition to FACS, a newly developed fluorescence image processing system (FIPS) will be used to measure fluorescence in frozen sections of tumors of spheroids, thus providing the capability of 3-dimensional reconstruction of the location of hypoxic tumor cells.